This invention pertains to transgenic cotton plants, plant material and seeds, characterized by harboring a specific transformation event, particularly by the presence of a gene encoding a protein that confers herbicide tolerance, at a specific location in the cotton genome. The cotton plants of the invention combine the herbicide tolerant phenotype with an agronomic performance, genetic stability and adaptability to different genetic backgrounds equivalent to the non-transformed cotton line in the absence of weed pressure.
All documents cited herein are hereby incorporated herein by reference.
The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps, which include extensive genetic characterization, breeding, and evaluation in field trials.
Cotton fiber is the single most important textile worldwide. About 80 million acres of cotton are harvested annually across the globe. Cotton is the fifth largest crop in the U.S. in terms of acreage production, with over 15 million acres planted in 2000. Primary weed species for cotton are Ipomoea sp. (morning glory), Amaranthus spp. (pigweed), Cyperus spp. (nutsedge), Xanthium spp. (cocklebur) and Sorghum spp. (johnsongrass). Before the introduction of broad-leaf herbicides that could be used on a growing cotton field, growers used directed, post-emergence applications of nonselective herbicides taking care not to contact the growing crop plants. As this requires a difference in height between the weeds and the crop, this is not always possible. Especially for small cotton, this practice is time-consuming and potentially damaging to the crop.
The bar gene (Thompson et al, 1987, EMBO J 6:2519-2523; Deblock et al. 1987, EMBO J. 6:2513-2518) is a gene encoding the enzyme phosphinothricin acetyl transferase (PAT), which, when expressed in a plant, confers resistance to the herbicidal compounds phosphinothricin (also called glufosinate) or bialaphos (see also for example U.S. Pat. Nos. 5,646,024 and 5,561,236) and salts and optical isomers thereof. Phosphinothricin controls broadleaf weeds including morning glory and has a wide window of application.
Successful genetic transformation of cotton has been obtained by a number of methods including Agrobacterium infection of cotton explants (Firoozabady et al. 1987, Plant Molecular Biology 10:105-116; Umbeck et al. 1987, Bio/Technology 5:263-266 and in WO 00/71733, U.S. Pat. No. 5,004,863, and U.S. Pat. No. 5,159,135), as well as direct gene transfer by microprojectile bombardment of meristematic cotton tissues (Finer and Mc Mullen, 1990, Plant Cell Reports, 5:586-589; McCabe and Martinell, 1993, Bio/Technology 11:596-598, WO92/15675, EP0 531 506). Increased transformation efficiency for Agrobacterium transformation has been reported using the methods described by Hansen et al. (1994, Proc. Nat. Acad. Sci. 91:7603-7607) Veluthambi et al. (1989, Journal of Bacteriology 171:3696-3703) and WO 00/71733.
Different methods for regeneration of cotton plants have also been described (WO 89/05344, U.S. Pat. No. 5,244,802, U.S. Pat. No. 5,583,036, WO89/12102, WO98/15622, and WO97/12512).
However, the foregoing documents fail to teach or suggest the present invention.
The present invention relates to a transgenic cotton plant, or seed, cells or tissues thereof, comprising, stably integrated into its genome, an expression cassette which comprises a herbicide tolerance gene comprising the coding sequence of the bar gene (as described in Example 1.1 herein), which is herbicide tolerant and, in the absence of weed pressure, has an agronomic performance which is substantially equivalent to the non-transgenic isoline. Under weed pressure and the appropriate Liberty(trademark) treatment, the plant will have a superior agronomic phenotype compared to the non-transgenic plant.
In one embodiment of the invention, the cotton plant or seed, cells or tissues thereof, comprises the expression cassette of pGSV71 (as described in Example 1.1, Table 1 herein). In the preferred embodiment of the invention the cotton plant or seed, cells or tissues thereof comprise elite event EE-GH1.
In another embodiment of the invention, the transgenic cotton plant or seed, cells or tissues thereof comprises:
(i) event EE-GH1 in its genome ;or
(ii) event EE-GH1 with the proviso that the bar gene used in the event is substituted with a nucleic acid sequence that hybridizes to the complement of the bar gene under stringent conditions.
More specifically, the present invention relates to a transgenic cotton plant, seed, cells or tissues thereof, the genomic DNA of which is characterized by the fact that, when analyzed in a PCR identification protocol as described herein, using two primers directed to the 5xe2x80x2 or 3xe2x80x2 flanking region of EE-GH1 and the foreign DNA, respectively, yields a fragment which is specific for EE-GH1. Preferably the primers are directed against the 5xe2x80x2 flanking region within SEQ ID NO: 1 and the foreign DNA respectively; most preferably, the primers comprise the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and yield a DNA fragment of between 250 and 290 bp, preferably of about 269 bp.
Reference seed comprising the elite event of the invention has been deposited at the ATCC under accession number PTA-3343. Thus, a preferred embodiment of the invention is the seed comprising elite event EE-GH1 deposited as ATTC accession number PTA-3343, which will grow into a cotton plant resistant to glufosinate. The seed of ATCC deposit number PTA-3343, which is a seed lot consisting of about 50% non-transgenic kernels and 50% transgenic kernels hemizygous for the transgene, comprising the elite event of the invention, which will grow into glufosinate tolerant plants. The seed can be sown and the growing plants can be treated with PPT or Liberty(trademark) as described herein to obtain 100% glufosinate tolerant plants, comprising the elite event of the invention. The invention further relates to cells, tissues, progeny, and descendants from a plant comprising the elite event of the invention grown from the seed deposited at the ATCC having accession number PTA-3343. The invention further relates to plants obtainable by propagation of and/or breeding with a cotton plant comprising the elite event of the invention grown from the seed deposited at the ATCC having accession number PTA-3343.
The invention further relates to plants, seeds, cells or tissues comprising a foreign DNA sequence, preferably a herbicide tolerance gene as described herein, integrated into the chromosomal DNA in a region which comprises the plant DNA sequence of SEQ ID NO: 1 and/or SEQ ID NO: 4, more particularly which comprises the DNA sequence of SEQ ID NO: 5, or a sequence which hybridizes under stringent conditions to a sequence that is complementary to a sequence comprising the plant DNA sequence of SEQ ID NO: 1, SEQ ID NO: 4 and/or SEQ ID NO: 5.
The invention further provides a process for producing a transgenic cell of a cotton plant, which comprises inserting a recombinant DNA molecule into a region of the chromosomal DNA of a cotton cell, tissue or callus which comprises the plant DNA sequence of SEQ ID NO: 1 and/or SEQ ID NO: 4, more particularly which comprises the DNA sequence of SEQ ID NO: 5, or which comprises a sequence which hybridizes under stringent conditions to a sequence that is complementary to a sequence comprising the plant DNA sequence of SEQ ID NO: 1, SEQ ID NO: 4 and/or SEQ ID NO: 5.
The invention further relates to a method for identifying a transgenic plant, or cells or tissues thereof, comprising elite event EE-GH1 which method is based on identifying the presence of characterizing DNA sequences or amino acids encoded by such DNA sequences in the transgenic plant, cells or tissues.
According to one preferred aspect of the invention, the method for identifying a transgenic plant, or cells or tissues thereof, comprising elite event EE-GH1, comprises amplifying a sequence of a nucleic acid present in biological samples, using a polymerase chain reaction, with at least two primers, one of which recognizes the plant DNA in the 5xe2x80x2 or 3xe2x80x2 flanking region of EE-GH1, the other which recognizes a sequence within the foreign DNA. Preferably, the genomic DNA is analyzed using primers which recognize a sequence within the plant 5xe2x80x2 flanking region of EE-GH1, most preferably within the plant DNA sequence in SEQ ID NO: 1, and a sequence within the foreign DNA, respectively. Especially preferably, the genomic DNA is analyzed according to the PCR identification protocol described herein whereby the primer recognizing a sequence within the 5xe2x80x2 flanking region comprises the nucleotide sequence of SEQ ID NO: 2.
Particularly, the primer recognizing a sequence within the 5xe2x80x2 flanking region comprises the nucleotide sequence of SEQ ID NO: 2 and the primer recognizing a sequence within the foreign DNA comprises the nucleotide sequence of SEQ ID NO: 3, so that the amplified fragment is a fragment preferably of between 250 and 290 bp, preferably of about 269 bp.
Accordingly, the present invention relates to the transgenic plant, cells or tissues thereof which can be identified according the above-described identification method for EE-GH1.
The present invention relates to methods for identifying elite event EE-GH1 in biological samples, which methods are based on primers or probes that specifically recognize the 5xe2x80x2 and/or 3xe2x80x2 flanking sequence of EE-GH1. In a preferred embodiment of the invention these methods are based on primers or probes which recognize a sequence within SEQ ID NO: 1 and/or SEQ ID NO: 4, more particularly primers or probes comprising the sequence of SEQ ID NO: 2.
The present invention further relates to the specific flanking sequences of EE-GH1 described herein, which can be used to develop specific identification methods for EE-GH1 in biological samples. More particularly, the invention relates to the 5xe2x80x2 and or 3xe2x80x2 flanking regions of EE-GH1, which can be used for the development of specific primers and probes as well as to the specific primers and probes developed from the 5xe2x80x2 and/or 3xe2x80x2 flanking sequences of EE-GH1. The invention further relates to identification methods for the presence of EE-GH1 in biological samples based on the use of such specific primers or probes.
The invention thus also relates to a kit for identifying elite event EE-GH1 in biological samples, the kit comprising at least one primer or probe which specifically recognizes the 5xe2x80x2 or 3xe2x80x2 flanking region of EE-GH1.
The invention also relates to a kit for identifying elite event EE-GH1 in biological samples, which kit comprises at least one specific primer or probe having a sequence which corresponds (or is complementary to) a sequence that hybridizes under stringent conditions to a specific region of EE-GH1. Preferably the sequence of the probe corresponds to a specific region comprising part of the 5xe2x80x2 or 3xe2x80x2 flanking region of EE-GH1. Most preferably the specific probe has (or is complementary to) a sequence that hybridizes under stringent conditions to the plant DNA sequence within SEQ ID NO: 1 or SEQ ID NO: 4.
Preferably the kit of the invention comprises, in addition to a primer which specifically recognizes the 5xe2x80x2 or 3xe2x80x2 flanking region of EE-GH1, a second primer which specifically recognizes a sequence within the foreign DNA of EE-GH1, for use in a PCR identification protocol. Preferably, the kit of the invention comprises two (or more) specific primers, one of which recognizes a sequence within the 5xe2x80x2 flanking region of EE-GH1, most preferably a sequence within the plant DNA region of SEQ ID NO: 1, and an other which recognizes a sequence within the foreign DNA. Especially preferably, the primer recognizing the plant DNA sequence within 5xe2x80x2 flanking region comprises the nucleotide sequence of SEQ ID NO: 2. Particularly, the primer recognizing the plant DNA sequence within 5xe2x80x2 flanking region comprises the nucleotide sequence of SEQ ID NO: 2 and the primer recognizing the foreign DNA comprises the nucleotide sequence of SEQ ID NO: 1 described herein.
The methods and kits encompassed by the present invention can be used for different purposes such as, but not limited to the following: to identify EE-GH1 in plants, plant material or in products such as, but not limited to food or feed products (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality (i.e. percentage pure material) of plant material comprising EE-GH1.
The present invention further relates to a method for tracking plants comprising elite event EE-GH1 in their genome upon introduction into different cultivars.
It will be understood that particular embodiments of the invention are described by the dependent claims cited herein.